Cytokines and polypeptide hormones bind to cell surface receptors and generate second messenger molecules, via signal transduction, that alter the metabolic direction of the cell. The receptor-ligand complexes are internalized by receptor-mediated endocytosis, and then localized to endosomes and lysosomes in the cytoplasm of the cell, followed by protease degradation. We postulated that the internalized ligand may have an intracellular role and tested this idea by directly microinjecting cytokines into the cytoplasmic compartment. Microinjection of IFNgamma induced Ia expression across the species barrier from an intracellular site and injection of TNFalpha induced rapid cytotoxicity and DNA fragmentation. TNFalpha treatment of JB6 cells induced the expression of novel form of the Jun transcription factor. The activity of this transcription factor correlates with cellular apoptosis and DNA fragmentation. TNFalpha-induced expression of Jun may program the cell to follow a specific metabolic pathway that results in cell death. A role for protein kinase (PKC), Ras, and phospholipase C (PLC) has been identified in the induction of macrophage surface Ia expression (a biological response seen normally from IFNgamma treatment) and apoptosis. These studies demonstrate a direct role for PKC, Ras, and PLC as intermediates in the biochemical pathways that lead to surface Ia expression. Injection of protein kinases A and phospholipase A2 had no effect on Ia expression. Thus, the PKC pathway can control expression of macrophage surface Ia, possibly by regulating the genes of the major histocompatibility complex and may play other roles in the activation of macrophages.